不同发育阶段表皮细胞微小RNA的差异表达谱分析

Different expression profiles of micro-RNA in epidermal cells of different developmental stages

目的探讨人表皮中具有未分化特性的表皮干细胞和角质形成细胞之间微小RNA（micro-RNA，miRNA）表达谱的差异。方法（1）采用酶消化法和Ⅳ型胶原快速贴壁相结合的方法获得人原代表皮干细胞及角质形成细胞。倒置显微镜下观察培养细胞的生长状况，免疫细胞化学染色法行B1整合素、角蛋白1（keratin1，CK1）、CK10、CK19单克隆抗体检测鉴定。（2）Trizol-步法分别提取表皮干细胞和角质形成细胞总RNA，甲醛变性胶电泳质检。mirVanaTMmiRNA分离试剂盒对其进行纯化，使用miRNA标记和杂交试剂盒进行荧光标记及芯片杂交，利用FeatureExtraction（V10．7）软件对杂交图片进行分析，GeneSpring（GX10．0）软件进行数据归-化及差异分析，同时应用RT-PCR法验证miRNA芯片结果的可靠性。（3）预测差异表达的miRNA的靶基因。结果（1）快速黏附于Ⅳ型胶原的细胞群培养3d能形成明显克隆，免疫细胞化学染色显示B1整合素及CK19呈阳性表达，为表皮干细胞；不能快速黏附于Ⅳ型胶原的细胞群培养3d无明显克隆形成，CK1及CK10呈阳性表达，为已分化角质形成细胞。（2）筛选出表皮干细胞中表达上调的nfiRNA31个，表达下调的miRNA153个。其中显著上调的miRNA有hsa-miRNA-125b-3p、hsa-miRNA-197-5P、hsa-miRNA-376a-3p等；显著下调的miRNA有hsa-miRNA-203、hsa-miRNA-29b-3p、hsa-miRNA-34a-3p等。其中上调的hsa-miRNA-197-5p和下调的hsa-miRNA-29b-3p的RT-PCR验证结果与芯片检测结果具有较好的-致性。（3）部分miRNA靶基因预测提示miRNA与细胞增殖分化、凋亡衰老等生物学特性有关。结论人表皮干细胞与角质形成细胞的miRNA表达存在明显差异，可能与两者不同的增殖分化能力等生物学特性有关。

Objective To investigate the difference in expression profiles of micro-RNA （miR- NA） between human epidermal stem cells and epidermal keratinoeytes. Methods （ 1 ） Human pri- mary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type IV collagen coated chosen method. Growth of cells cultured in vitro was obserced by inverted microscope. Monoclonal antibody of integrinl , keratin 1 （ CKI ） , CK10, and CK19 were detected by immunocyto- chemical staining. （2）Total RNA was respectively isolated from epidermal stem cells and epidermal kera- tinocytes by Trizol-based single-step procedure, detected by formaldehyde denaturing gel electrophoresis, purified by mirVanaTM miRNA isolation kit, and then labeled and hybridized by miRNA labeling and hy- bridization kit. Images of hybridization were analyzed using the Feature Extraction （Version 10.7）. Data normalization and difference analysis were performed with GeneSpring （ GX 10.0）. Moreover, miRNA microarray results were confirmed by RT-PCR. （3） Target genes of differently expressed miRNA were predicted. Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinl31 and C K19 were positive. Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive. （2） Of the epidermal stem cells, 31 miRNAs were up-regulated and 153 down-regulated. Besides, significantly up-regulated miRNAs were hsa-miRNA-125b-3p, hsa-miRNA- 197-5p, and hsa-miRNA-376a-3p, while significantly down-regulated miRNAs were hsa-miRNA-203, hsa-miRNA-29b-3p, and hsa-miRNA-34a-3p. Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR- 29b-3p revealed high concordance with the microarray results. （3） Some miRNAs target genes indicated that miRNA related to cell proliferation, differentiation, apoptosis, aging and other biological characteristics. Conclusion Distinct differences in miRNA expression profiles are detected between human epi- dermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability.